Iron sulfur clusters are viewed as the prototypical electron transfer cofactors, but there is increasing evidence in a number of systems that such clusters undergo proton-coupled electron transfer (PCET). In the mitochondria, for instance, several [2Fe-2S] clusters mediate PCET reactions, including Rieske clusters, which are involved with ATP synthesis, and mitoNEET clusters, which help regulate this process. Both of these clusters facilitate PCET, and proposed here are model studies to develop a detailed understanding of such processes. As part of the Q-cycle of the bc1 complex, Rieske clusters are involved in the redox cycling of quinones, a process that requires tight regulation to avoid formation of semiquinone intermediates which ultimately leads to oxidative stress and tissue damage. The mitoNEET protein has been identified as the target of the thiazolidinedione (TZD) family of drugs, which are used to treat type 2 diabetes, and this protein is a SRWHQWLDO WDUJHW IRU $O]KHLPHU6V 3DUNLQVRQ6V DQG VWURNH GUXJV Knowledge of how these and related clusters mediate PCET is key to understanding their biological function and could lay the foundation for new treatments. Owing to the complexity of the biological systems, this understanding is best developed with well- characterized model systems. The goals of the proposed experimental studies are: i) to prepare and study the PCET reactivity of synthetic [2Fe-2S] clusters that are models for Rieske and mitoNEET clusters, and ii) to establish factors that affect the rate and mechanism of PCET to/from [Fe-S] clusters. Biomimetic [2Fe-2S] clusters will be prepared in both biologically relevant oxidation states, FeIII/III and FeII/III, and in both their protonated and deprotonated states. These congeners will be thoroughly characterized by a variety of techniques, including NMR, IR, EPR, and UV-vis spectroscopy, SQUID magnetometry, and x-ray crystallography. The thermochemical properties of these clusters - their redox potentials and pKa values 1 will also be measured. Kinetic and mechanistic studies of PCET and ET reactions will then be undertaken, with an emphasis on biologically relevant reactions. Combining all these various measurements will lead to a detailed description of the PCET reactivity of such clusters, including, for example, how the extent of magnetic coupling between the two iron centers affects PCET. These systems will then be compared with other model [Fe-S] systems that do not contain an imidazole or imidazolate ligand. Protonation of these congeners will likely occur at the sulfur, providing an understanding of how the site of protonation affects PCET at [Fe-S] clusters. Collectively, these studies will provide a fundamental description of PCET at [Fe-S] clusters, and the results of these studies may provide insights to the above-mentioned diseases. Additionally, these results will guide future mechanistic proposals for enzymes that feature [Fe-S] clusters, and will further our understanding of PCET at sulfur and at multi-metallic sites, both of which are prevalent in biological systems. PUBLIC HEALTH RELEVANCE: The proposed research will provide a fundamental description of the proton-coupled electron transfer (PCET) reactions at biomimetic [2Fe-2S] clusters. Several proteins that incorporate [2Fe-2S] clusters mediate PCET, and malfunctions in the clusters have been associated with numerous diseases including mitochondrial myopathies and Wolfram Syndrome 2. Moreover, some of these proteins are potential targets for $O]KHLPHU6V 3DUNLQVRQ6V stroke, and type 2 diabetes therapeutics.